Reaction Mechanism of the Ca2+-dependent ATPase of Sarcoplasmic Reticulum from Skeletal Muscle

Abstract

The time course of Ca²⁺ uptake by the SR was compared with those of formation and decomposition of a phosphorylated intermediate, EP, in the Ca²⁺, Mg²⁺-depend-ent ATPase reaction [EC 3.6.1.3]. Ca²⁺ uptake was measured by the Millipore nitration method, using ⁴⁵CaCl₂ as a substrate. The SR was allowed to react with 10 μM ATP at 0°C in the presence of 20 μM CaCl₂ without adding MgCl₂. Under the conditions used, the formation and decomposition of EP could be measured separately, since the rate of EP formation was much higher than that of its decomposition. The following results were obtained. 1. When EP formation and Ca²⁺ uptake by the SR were terminated by adding 1 mM EGTA+2.5mM MgCl₂, EP was rapidly decomposed into E+Pi. Ca²⁺ uptake consisted of two phases: a rapid, initial one and a slow, steady one, which was almost linear with time. The time course of the former corresponded to that of EP formation, and that of the latter to P₁ liberation before the addition of EGTA+MgCl₂. During both phases, the molar ratio of Ca²⁺ uptake/ATP hydrolysis was about 1/1. 2. When the reaction was stopped by adding lmM EGTA+0.25mM ADP, ATP was formed immediately from EP and ADP. The time course of Ca²⁺ uptake showed only a slow, steady phase, corresponding to that of P₁ liberation before the addition of EGTA+ADP. The molar ratio of Ca²⁺ uptake/ATP hydrolysis was smaller than 1/1. 3. After Ca²⁺ uptake had been stopped by adding lmM EGTA+1OμM ADP, the time course of release of Ca²⁺ ions from the SR was measured by adding 20 mM MgCU after various intervals, since the release was stopped by adding MgCl₂. The time course observed corresponded to that of disappearance of EP.